. The investigators have developed a laboratory system for the directed evolution of RNA enzymes. They propose to use this system to develop ribozymes that are best able to cleave target sites within HIV-1 RNA or DNA, optimizing for activity under conditions that resemble those of the cellular environment. The optimization procedure is carried in vitro, employing a population of well over a thousand billion mutant ribozymes that are subjected to repeated rounds of selective amplification.Each round of selective amplification requires 1-2 days to perform, so that they can survey a very large number of ribozyme variants in a short period of time. The goal of the proposed research is to develop a family of anti-HIV-1 ribozymes through the use of in vitro evolution methodology. The applicants are especially interested in developing ribozymes that cleave double-stranded DNA with high specificity and selectivity. This will be done by modifying a group I ribozyme so that it binds to doubled- stranded DNA through triple-helical interactions, and then carrying out in vitro evolution using a series of DNA substrates that have progressively more stable duplex structure. DNA-cleaving group I ribozymes will be targeted against the integrase recognition sequence at the U5 end of HIV-1 DNA and against the gene encoding the Tat regulatory protein. They will develop group II ribozymes as well, focusing on the RNA cleavage reaction for which they are likely to be more discriminating than group I ribozymes. RNA-cleaving group II ribozymes will be targeted against a highly-conserved region within tat mRNA. Finally they will attempt to exploit the ability of E. coli single-strand binding protein to trans-activate a DNA-cleaving group I ribozyme, leading to the development of anti-HIV-1 nucleoproteins.